This invention was made in the course of work supported in part by the United States Government, and the Government has certain rights in the invention.
This invention relates to isolation, identification and therapeutic use of populations of T cell lymphocytes, and particularly of populations of T cell lymphocytes that are capable of binding to specific extracellular matrix proteins.
The term "leukocyte" refers to any of the nucleated cells normally present in blood or tissues whose major function is defense against foreign invaders of the body. Our understanding today regarding the different types of leukocytes stems from the classic staining researches of Paul Ehrlich beginning in 1878 whose discovery of polychromatic and supravital stains led to the :first important advances in the knowledge of leukocytes. His pioneer investigations based on the morphology of the different leukocytes coupled with the first understanding of the maturation sequence via the work of Pappenhein in 1914 led directly to the classification system used for decades thereafter. Initially, the different types of leukocytes were separated according to: (1) the type of defense function provided--i.e., phagocytosis, antibody production, or cellular immunity; (2) the shape of the nucleus of the cell--i.e., polymorphonuclear or mononuclear; (3) the site of origin for the cell--i.e., myeloid or lymphoid; and (4) the presence or absence of intracellular specific staining granules--i.e., granulocytes or non-granulocytes. This led directly to the identification and establishment of normal values of different types of leukocytes as neutrophils, eosinophils, basophils, monocytes (including macrophages), and lymphocytes. Of these five major leukocyte types, the lymphocyte (although first defined as a morphological entity in 1879) has since the early 1950's been the subject of major, intense, clinical, and research investigations which have led to unparalleled advances in our understanding of how these cells are created, organized, and function in vivo.
Lymphocytes are mononuclear cells whose cytoplasm does not contain any specific staining granules. As with other leukocytes and red blood cells, they originate in the bone marrow. Lymphocytes acquire their immunocompetence in the bone marrow and thymus, and reside in the organs of the peripheral lymphoid system (lymph nodes, spleen, adenoids and tonsils, the Peyer's patches of the small intestine) where they encounter antigens and mount immune responses. They also circulate in the peripheral blood; and may recirculate between the blood stream, the lymphoid organs, and sites of immune reactions in the tissues.
Lymphocytes have been divided broadly into two major orders: B-cells responsible for antibody production and the antibody (or humoral) immune response; and T cells responsible for cellular immune responses and immune regulation generally. In recent years the T cell lymphocyte has become ever more intensively explored.
In general, the present state of knowledge and understanding regarding T cell lymphocytes is based upon three different and complementary investigational approaches. These are, in summary: (a) study of the functional properties of T lymphocyte subsets; (b) study of the surface antigens found on T cells and their subsets, using specific monoclonal antibodies; and (c) use of the methods of biochemistry and molecular biology to investigate mechanisms of T cell functional activities and specificity. Because of the complexity of the immune system and the newness and rapidly changing nature of these modern immunological studies (e.g., most of the monoclonal antibodies which have made these studies possible were not available before the early 1980's), the results of these various studies can be difficult to understand and correlate. Therefore, the three approaches are discussed in greater detail below.
The first investigational basis is the functional designation resulting from an empirically observed in vivo and/or in vitro specific biological activity. Such observations provide functional designations including: helper/inducer T cell, cytotoxic/suppressor T cell, lymphokine activated killer cell, tumor infiltrating lymphocyte cell, delayed hypersensitivity cell, dendritic epidermal T cell, and intraepithelial lymphocyte (the latter two terms designating T cell subsets that can localize in particular areas of the body). Each of these describes an observed function of lymphocytes. The designations employed are solely in functional or operative terms because each identification is based exclusively on the observed capability or attributes of one type of T cell able to participate in specific biological activities and/or immunological events.
The second basis of T cell lymphocyte classification resulted from the ability to produce specific antibodies which are then employed to characterize and define the surface antigenic determinants or cell surface markers found on different kinds of T cell lymphocytes. With the ability to produce monoclonal antibodies such as the OKT series (produced by Ortho Diagnostic System, Inc.), the Leu series (produced by Beckon-Dickinson), and the Coulter series (produced by Coulter Immunology), the individual T cell antigenic designations for man, mouse, and other animals were created. In order to bring some order to the confusing alternative nomenclatures which have resulted from the development of various sets of monoclonal antibodies provided by different laboratories, a new nomenclature system was adopted by the First International Workshop on Human Leukocyte Differentiation Antigens (Jour. Immunol. 134:659-660 (1985); see also Knapp et al., Immunol. Today 10:253-258 (1989)]. Using this nomenclature system, all monoclonal antibodies that appear to detect a particular antigen are assigned to a numbered "cluster of differentiation" or "CD" for that antigen. It has been found that particular T cell subsets initially defined by function (such as helper and cytotoxic/suppressor T cells) also have characteristic cell surface markers as defined by specific monoclonal antibodies. The biochemical function of these cell surface markers is itself an active area of investigation. The phenotyping of the different T cells and their separation into different subclasses based on their individual surface markers has become the most favored investigational technique among researchers today.
The third investigational basis for identifying and distinguishing differences among the various populations comprising T cell lymphocytes involves molecular biological and molecular genetic studies of proteins involved in immunological activities of T cells, including recognition and responses to specific antigens. The focus of these studies is the T cell receptor which is responsible for the recognition of a specific antigen by that individual T cell.
T cell receptors ("TCR") on the classically defined types of cells such as helper and cytotoxic T cells were found to be composed of two subunits termed "TCR alpha and beta" proteins. These proteins are the specific products of individual genes that are themselves rearranged during thymic ontogeny (see, e.g., Allison et al., Jour. Immunol. 192:2293-2300 (1982); Meuer et al., Jour. Exp. Med. 157:705-719 (1983); Haskins et al., Jour. Exp. Med. 157:1149-1169 (1983)). These TCR alpha/beta (".alpha..beta.") protein molecules are found to comodulate, to coimmunoprecipitate, and to be coexpressed with the CD3 glycoprotein; and the direct physical association of the two protein complexes was demonstrated by chemically cross-linking the TCR .alpha..beta. molecules to the CD3 glycoproteins. These .alpha..beta. cell receptors do not recognize soluble antigens. Rather, they recognize antigens on cell surfaces, complexed with .proteins of the major histocompatibility complex (or "MHC"). Moreover, the MHC protein must be of the correct "self" type (i.e., an MHC protein from the same individual organism or an organism genetically identical at that locus) as the T cell and not from a genetically different individual. The recognition of such antigens is thus said to be "MHC restricted". Helper T cells recognize antigens coupled with one class ("Class II") of MHC proteins on the surface of antigen-presenting cells such as macrophages; and the surface protein CD4 on the surface of these T cells is thought to serve as an accessory binding factor for the MHC protein. Cytotoxic T lymphocytes (or "CTL") recognize the surface antigens of their target cells when complexed with another class ("Class I") of the MHC proteins also found on the surface of the target cell. The surface protein CD8 on the CTL surface again is thought to serve as an accessory binding factor. Recognition of antigen results in a cascade of events within the T cell which leads to the expression of helper or cytotoxic functions. In the case of cytotoxicity, the CTL lyses its specific target cells. (For a general review of these processes, see the textbook by J. W. Kimball, "Introduction to Immunology," Second Edition, New York, MacMillan Publishing Company, 1986.)
More recently however, there has been an identification of a second T cell receptor protein complex directly associated and controlled by a different third and fourth gene designated gamma/delta (or ".gamma..delta.") genes. These .gamma..delta. genes have been identified in the mouse and in man, and cells bearing .gamma..delta. TCRs constitute between 1 and 10% of peripheral T cells in both humans and mice. The present state of knowledge regarding the class of T cells carrying TCRs which are encoded by the .gamma..delta. genes is documented by the following representative publications: Brenner et al., Nature 322:145-149 (1986); Brenner et al., Nature 325:689-694 (1987); Faure et al., Jour. Immunol. 141:3357-3360 (1988); Janeway et al., Immunol. Today 9:73-76 (1988); Bluestone, J. and L. A. Matis, Jour. Immunol. 142:1785-1788 (1989); Band et al. Science 238:682-684 (1987); Janeway, C.A., Nature 333:804-806 (1988); and Hercend, T. and R. E. Schmidt, Immunol. Today 2:291-293 (1988).
In short, it is now clear that two lineages of T cells bearing the CD3 antigenic complex can be defined based upon the biochemical nature and presence of the heterodimeric receptor chains expressed as either .alpha..beta. proteins or .gamma..delta. proteins. The TCR .alpha..beta.T cells include the majority classes of T cells with classic MHC-restricted helper, cytotoxic, and suppressor activities.
The function (or functions) of .gamma..delta. T cells is unknown. Gamma/delta T cells have been widely demonstrated to carry out non-MHC-restricted cytotoxic activity against a variety of tumor cells. However, this requires high concentrations of interleukin-2; and appears to be a lymphokine activated killer (or "LAK") phenomenon, a condition which can be demonstrated by .alpha..beta.T cells as well (see, e.g., Matis et al., Nature 330:262 (1987); Brooks, Nature 305:155 (1983); Shortman et al., Curr. Topics. Microbiol. Immunol. 116:111 (1986); Maziarz et al., Seventh Int. Congress Immunol., 1989, Abstract). Thus, the non-MHC-restricted cytotoxicity demonstrated by .gamma..delta. T cells may not be the usual physiological function(s) of these cells. Recent studies have demonstrated that at least some .gamma..delta. T cells can recognize MHC molecules; and in at least one case, recognize a specific antigen in an MHC-restricted manner (Bluestone et al., Jour. Exp. Med. 168:1899 (1988); Kozbor et al., Jour. Exp. Med. 169:1847 (1989)). In another laboratory, .gamma..delta. T cells have been isolated from the peripheral blood of patients with B cell lymphomas that can lyse their specific B cell lymphomas in vitro.
A particularly noteworthy feature of .gamma..delta. T cells is their localization in tissues. In the mouse, particular narrow classes of .gamma..delta. T cells have been shown to localize in the skin and intestinal epithelia (Asarnow et al., Cell 55:837 (1988); Goodman and Lefrancois, Nature 333:855 (1988)). No such localization in normal tissues has been found in the human (Groh et al., Jour. Exp. Med. 169:1277 (1989)); however, human .gamma..delta. T cells do appear to localize in various inflammatory sites. In the case of the human gut, although normal levels of .gamma..delta. T cells have been reported on the average to be low, they have also been reported to be significantly higher (17-33%) in the intestines of children with celiac disease (Russell et al., FASEB Jour. 3:A485 (1989); Spencer and Isaakson (letter) Nature 337:416 (1989)). Gamma/delta T cells have been found in numbers somewhat higher than in blood in the synovial fluid and membranes of adult rheumatoid arthritis patients (Brennan et al., Jour. Autoimmunity 1:319 (1988)). Moreover, clones of .gamma..delta. T cells have been isolated from cerebrospinal fluid of patients with subacute sclerosing panencephalitis (Anget al., Jour. Exp. Med. 165:1453-1458 (1987)) and from joint fluid of patients with juvenile rheumatoid arthritis (DeMaria et al., Eur. Jour. Immunol. 17:1815-1819 (1987)). In both humans and mice, .gamma..delta. T cells have been found to localize at anatomically distinct regions of the lymphoid system (Bucy et al., Jour. Immunol. 1412:2200 (1988)). In contrast, .alpha..beta. T cells constitute the bulk of the T cell populations in peripheral blood and peripheral lymph nodes, while .gamma..delta. T cells comprise at most a few percent. The basis of the ability of .gamma..delta. T cells to localize within tissues is at present unknown.
The true biological function and capability of TCR .gamma..delta. cells remains effectively unknown--has been explicitly noted in the literature (Bluestone, J. and L. A. Matis, Jour. Immunol. 142:1785-1788 (1989)). In addition, although great strides have been made in the last few years by defining the TCR .gamma..delta. cells in terms of their tissue distribution and protein receptor/gene organization format, the true physiological role and the potential receptor ligand for such TCR .gamma..delta. cells remains elusive. Although much speculation and hope is placed upon future research investigations for TCR .gamma..delta. cell populations, there is a continuing ignorance regarding methods for isolating such TCR .gamma..delta. cells without using flow cytometry techniques and apparatus; and a continuing absence of knowledge or understanding of the attributes or potential uses and applications for individual populations of TCR .gamma..delta. cells.